Accelerated Genital Tract Aging in HIV: Estradiol Clinical Trial

HIV may be associated with premature aging in the female genital tract including alterations in the vaginal microbiome and mucosal inflammation, which may increase risk for vaginal atrophy, urinary tract infections (UTI) and other genital tract infections. This study will determine whether use of vaginal estradiol for 12 weeks in menopausal women living with HIV with symptomatic vaginal atrophy will improve atrophy symptoms and the vaginal microbiome and reduce mucosal inflammation thereby improving vaginal health. This study will include 50 participants randomized to treatment with a vaginal estradiol insert or no therapy for 12 weeks and will have 4 study visits.

• Estradiol Vaginal Insert (Drug), also known as Vagifem®, Vagifem (estradiol vaginal tablet): Using a pre-loaded single-use plastic applicator, participants will insert one 10 microgram estradiol tablet intravaginally daily for 2 weeks and then one tablet twice weekly for the remainder of the study for a total of 12 weeks.

Primary outcomes

• Change in Most Bothersome Symptom (MBS) of Vaginal Atrophy [Between baseline (Visit 2) and 12 weeks (Visit 5)]: Change in the severity of MBS of vaginal atrophy as reported during the baseline visit was assessed at 12 weeks (Visit 5). During the baseline visit, participants were asked to identify their MBS and assess the severity of the MBS on an ordinal scale of "None," "Mild," "Moderate," or "Severe." During the follow-up visit at 12 weeks participants were again asked to identify and assess the severity of their MBS. The degree of severity of the MBS reported at baseline was then compared to the severity of the MBS reported at 12 weeks and categorically summarized and reported as either "Severity Increased" "Severity Decreased" or "No change in Severity" for the given MBS. Participants whose MBS reported at baseline changed during the follow-up visit at 12 weeks were excluded from the analysis. Participants who did not report an MBS during the baseline visit were also excluded. Data for each possible type of MBS is summarized by study arm.
• Vaginal Microbiome - Relative Abundance of Lactobacillus Crispatus (L. Crispatus) [Between baseline (Visit 2) and 6 weeks and 12 weeks (Visit 5)]: The relative abundance of the protective Lactobacillus species, L. crispatus, as quantified by lllumina MiSeq sequencing will be calculated by dividing the total number of L crispatus sequences detected in a sample by the total number of sequences from all bacterial species detected in the same sample. This proportion will be expressed as a percentage. The change in relative abundance between baseline (visit 2) and 12 weeks (visit 5) will be summarized by study arm.
• Vaginal Microbiome - Quantitative Determination of Protective Lactobacilli Species [Between baseline (Visit 2) and 6 weeks and 12 weeks (Visit 5)]: Changes in the vaginal microbiome, specifically the quantities of protective Lactobacilli species (L. crispatus, L. jensenii and L. gasseri) as measured by quantitative PCR (qPCR) will be determined. The three Lactobacilli species will be identified and quantified in colony forming units per milliliter of sample (CFU/mL). Changes in abundance from baseline will be summarized by study arm using basic descriptive statistics.

Secondary outcomes

• Vaginal Microbiome - Relative Abundance of Bacterial Vaginosis Associated Species [Between baseline (Visit 2) and 6 weeks and 12 weeks (Visit 5)]: The relative abundance of bacterial vaginosis (BV) associated bacterial species as quantified by lllumina MiSeq sequencing, will be calculated by dividing the total number of the individual BV-associated species sequences in a sample by the total number of sequences from all bacterial species detected in the same sample. This proportion will be expressed as a percentage. The change in relative abundance between baseline (visit 2) and 12 weeks (visit 5) will be summarized by study arm for the 3 most common BV-associated species; Gardnerella vaginalis (G. vaginalis); Fannyhessea vaginae (F. vaginae); and Prevotella bivia (P. bivia)
• Vaginal Microbiome - Quantitative Determination of Bacterial Vaginosis Associated Species [Between baseline (Visit 2) and 6 weeks and 12 weeks (Visit 5)]: Changes in quantities of BV-associated species as measured by quantitative PCR (qPCR) will be determined. BV-associated species will be detected and quantified in colony forming units per milliliter of sample (CFU/mL). Changes in abundance from baseline for each detected species will be summarized by study arm using basic descriptive statistics.
• Change in Vaginal Cytokine and Chemokine Concentrations [Between baseline (Visit 2) and 6 weeks and 12 weeks (Visit 5)]: Change in concentrations from baseline of vaginal cytokines and chemokines in cervicovaginal lavage (CVL) was determined. Following assay, concentrations for the following cytokines and chemokines, as individually expressed, were reported in picograms per milliliter (pg/mL): IL-1A, Interleukin-8 (IL8); Interferon-gamma inducible protein 10 (IP-10); Monocyte Chemoattractant Protein-1 (MCP-1); and Secretory Leukocyte Protease Inhibitor (SLPI). Change in concentrations for the respective cytokines and chemokines from baseline are summarized by study arm using basic descriptive statistics.

Other outcomes

• HIV-1 RNA Levels in the Genital Tract [Baseline and 6 and 12 weeks]: HIV-1 RNA testing from cervicovaginal lavage fluid will be used to assess HIV-1 concentrations in the genital tract. Concentrations will be summarized by study arm and subsequently analyzed by multivariate linear regression. Cervicovaginal lavage (CVL) fluid was collected for other measures however due to insufficient funding, CVL HIV-1 viral loads were not run at any time point therefore the data is not available, nor will it be available in the future.
• Immunoglobulin (Ig)A and IgG Coated Bacteria [Baseline and 6 and 12 weeks]: Relative differences in levels of live IgA+IgG+ coated, live IgA+IgG- coated, live IgA-IgG-coated and dead bacteria will be summarized using basic descriptive statistics and subsequently analyzed by multivariate linear regression. Vaginal swabs were collected for quantification of subsets of IgA and IgG coated bacteria however due to insufficient funding, sequencing of these samples has not been performed and therefore data is not available. If we are able to secure additional funding, the sequencing may be performed in the future.

Sex: Female, ages: 45 Years to 70 Years, accepts healthy volunteers

Inclusion Criteria:

* HIV infection
* Females aged 45-70
* Menopause defined by having no menstrual periods for 12 consecutive months, confirmed with serum follicle-stimulating hormone (FSH) level >40 IU/ml and serum estradiol level <20 pg/ml
* Symptomatic vaginal atrophy defined as reporting at least once per week in the past 30 days, 1 or more of the following symptoms of moderate or severe intensity: Dryness, Itching, Irritation, Soreness or pain OR Pain associated with sexual activity at least once
* Evidence of atrophy on exam, including thin, pale and dry vaginal and vulvar surfaces
* Agrees not to use vaginal products other than vaginal estradiol tablet during the clinical trial

Exclusion Criteria:

* Current or previous history of breast cancer or estrogen dependent neoplasia
* Current or past thromboembolic disease (deep vein thrombosis or pulmonary embolism, not including thrombophlebitis)
* Current or previous history of myocardial infarction or stroke
* Known blood clotting disorders including Protein C, Protein S and antithrombin deficiency, Factor V Leiden or prothrombin mutations
* Known severe liver disease including cirrhosis or active Hepatitis B
* History of adverse reaction to vaginal estradiol
* Current unexplained or unevaluated abnormal genital bleeding
* Current or suspected pregnancy
* If < age 55, had a hysterectomy and has at least one ovary
* Pelvic or vaginal surgery in the prior 60 days
* Use of systemic reproductive hormones in the past 2 months
* Antibiotic use in the past 30 days
* Use of immunosuppressive medications in the prior 60 days including biologics, chemotherapeutics or post-transplant immunosuppressive medications
* Use of any vaginal or vulvar preparations 1 month prior to enrollment
* Current active vaginal infection (diagnosed by wet mount at Visit 1 or 2)
* Any serious disease or chronic condition that might interfere with study compliance
* Unwilling to agree to the provisions of the protocol

Participant flow

Outcome results

• Change in Most Bothersome Symptom (MBS) of Vaginal Atrophy (Primary), Count of participants, Participants: Estradiol Vaginal Insert 0; No Treatment 1
• Vaginal Microbiome - Relative Abundance of Lactobacillus Crispatus (L. Crispatus) (Primary), Mean, change in percentage of sequences: Estradiol Vaginal Insert -1.37 +/- 4.25; No Treatment -6.9 +/- 25.14
• Vaginal Microbiome - Quantitative Determination of Protective Lactobacilli Species (Primary)
• Vaginal Microbiome - Relative Abundance of Bacterial Vaginosis Associated Species (Secondary), Mean, change in percentage of sequences: Estradiol Vaginal Insert 3.15 +/- 27.69; No Treatment -1.22 +/- 17.03
• Vaginal Microbiome - Quantitative Determination of Bacterial Vaginosis Associated Species (Secondary)
• Change in Vaginal Cytokine and Chemokine Concentrations (Secondary), Mean, pg/mL: Estradiol Vaginal Insert 0.17 +/- 0.53; No Treatment -0.07 +/- 0.14
• HIV-1 RNA Levels in the Genital Tract (Other pre specified)
• Immunoglobulin (Ig)A and IgG Coated Bacteria (Other pre specified)
• Change in Vaginal Symptom Index (VSI) (Post hoc), Mean, score on a scale: Estradiol Vaginal Insert -1.01 +/- 0.65; No Treatment -0.38 +/- 0.62
• Vaginal Microbiome - Detectable Lactobacillus Crispatus (L. Crispatus) (Post hoc), Count of participants, Participants: Estradiol Vaginal Insert 3; No Treatment 5
• Vaginal Microbiome - Detectable Bacterial Vaginosis-Associated Species (Post hoc), Count of participants, Participants: Estradiol Vaginal Insert 11; No Treatment 16
• Change in Vaginal Microbiome - Detectable Lactobacillus Crispatus (L. Crispatus) (Post hoc), Number, percentage change detectable sequences: Estradiol Vaginal Insert -9.1; No Treatment -8.0
• Change in Vaginal Microbiome - Detectable Bacterial Vaginosis-Associated Species (Post hoc), Number, percentage change detectable sequences: Estradiol Vaginal Insert -13.6; No Treatment 8.0

Adverse events

Time frame: Up to 13 weeks following randomization

Serious adverse events by term

Other adverse events by term

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• Study Protocol and Statistical Analysis Plan (PDF, 2021-09-09)
• Informed Consent Form (PDF, 2023-06-14)

• Kerry J Murphy, MD, Principal investigator, Albert Einstein College of Medicine

NIH grants: K23AG062400, 2019